The cyclization of the universal precursor geranyl diphosphate (GPP) to form monocyclic and bicyclic monoterpenes is catalyzed by a group of enzymes termed monoterpene synthases (or cyclases). The biochemical transformation of GPP to cyclic products has been investigated using enzymes from a variety of plants, including both angiosperms (Croteau, R., Chem. Rev. 87:929-954, 1987) and gymnosperms (Lewinsohn et at., Arch. Biochem. Biophys. 293:167-173, 1992; Savage et al., i J. Biol. Chem. 269:4012-4020, 1994; Savage et al., Arch. Biochem. Biophys. 320:257-265, 1995). A mechanistic paradigm for these transformations is well established (Croteau, R., Chem. Rev. 87:929-954, 1987; Wise, M. L., and Croteau, R., in Comprehensive Natural Products Chemistry: Isoprenoids (Cane, D. E., ed) Vol. 2 (in press), Elsevier Science, Oxford, 1998). In summary, geranyl diphosphate is initially ionized and isomerized to form either 3R- or 3S-linalyl diphosphate, depending on the particular enzyme, which is converted to the .alpha.-terpinyl cation as a central intermediate. Further transformations of this reactive intermediate may be effected by additional intramolecular electrophilic additions, hydride shifts or other rearrangements before termination of the sequence by deprotonation of the final cation or capture by an external nucleophile, such as a hydroxyl ion or the diphosphate group. Although the fate of the substrate has been well characterized in numerous monoterpene cyclization reactions, the molecular mechanisms by which the enzymes effect these transformations is still poorly understood.
Culinary sage (Salvia officinalis) produces a number of monoterpenes, including (+)- and (-)-.alpha.-pinene, (+)- and (-)-.beta.-pinene, (+)- and (-)-camphene, (+)-sabinene, (+)- and (-)-limonene, myrcene, 1,8-cineole, and (+)-bornyl diphosphate (Croteau, R., Chem. Rev. 87:929-954, 1987). Because sage produces this broad range of acyclic, monocyclic and bicyclic monoterpenes, including several olefin isomers, a cyclic ether and a diphosphate ester, this plant has provided an ideal system for the study of a variety of biosynthetic enzymes, all of which utilize the same substrate but produce different products by variations on a single reaction mechanism (Croteau, R., Chem. Rev. 87:929-954, 1987; Wise, M. L., and Croteau, R., in Comprehensive Natural Products Chemistry: Isoprenoids (Cane, D. E., ed) Vol. 2 (in press), Elsevier Science, Oxford, 1998). These monoterpene synthases include (+)-bornyl diphosphate synthase (the enzyme producing the precursor of (+)-camphor) (Croteau, R., and Karp, F., Arch. Biochem. Biophys. 198:512-522, 1979; Croteau, R., and Karp, F., Arch. Biochem. Biophys. 198:523-532, 1979), 1,8-cineole synthase (Croteau et al, Arch. Biochem. Biophys. 309:184-192, 1994), (+)-sabinene synthase (the enzyme producing the precursor of (-)-3-isothujone) (Croteau, R., in Recent Developments in Flavor and Fragrance Chemistry (Hopp, R., and Mori, K., eds), pp. 263-273, VCH, Weinheim, Germany, 1992; Croteau, R., in Flavor Precursors: Thermal and Enzymatic Conversions (Teranishi, R., Takeoka, G. R., and Guntert, M., eds), American Chemical Society Symposium Series, No. 490, pp. 8-20, Washington, DC, 1992), and several pinene synthases (Gambliel, H., and Croteau, R., J. Biol. Chem. 257:2335-2342, 1982; Gambliel, H., and Croteau, R., J. Biol. Chem. 259:740-748, 1984; Wagschal et al., Arch. Biochem. Biophys. 308:477-487, 1994; Pyun et al., Arch. Biochem. Biophys. 308:488-496, 1994).
As is typical of monoterpene cyclases, many of these enzymes from sage generate multiple products from geranyl diphosphate (Wise, M. L., and Croteau, R., in Comprehensive Natural Products Chemistry: Isoprenoids (Cane, D. E., ed) Vol. 2 (in press), Elsevier Science, Oxford, 1998; Wagschal et al., Tetrahedron 47:5933-5944, 1991). For example, investigations with the partially purified native enzymes have suggested that a single enzyme, termed (+)-pinene synthase (cyclase I), is responsible for the synthesis of both (+)-.alpha.-pinene and (+)-camphene, with lesser amounts of (+)-limonene and myrcene, whereas a second enzyme, (-)-pinene synthase (cyclase II), has been shown to produce (-)-.alpha.-pinene, (-)-.beta.-pinene and (-)-camphene, with minor amounts of (-)-limonene, terpinolene and myrcene (Gambliel, H., and Croteau, R., J. Biol. Chem. 257:2335-2342, 1982; Gambliel, H., and Croteau, R., J. Biol. Chem. 259:740-748, 1984).
More recently, a third synthase from sage, termed cyclase III, has been described which produces a mixture of (+)-.alpha.-pinene and (+)-.beta.-pinene, along with minor amounts of myrcene (Wagschal et al., Arch. Biochem. Biophys. 308:477-487, 1994; Pyun et al., Arch. Biochem. Biophys. 308:488-496, 1994). Evidence that these reactions are catalyzed by individual, multifunctional enzymes is provided by co-purification and differential inhibition studies (Gambliel, H., and Croteau, R., J. Biol. Chem. 259:740-748, 1984), as well as by isotopically sensitive branching experiments (Wagschal et al., Arch. Biochem. Biophys. 308:477-487, 1994; Wagschal et al., Tetrahedron 47:5933-5944, 1991; Croteau et al., Biochemistry 26:5383-5389, 1987). In spite of considerable effort, the (+)-pinene synthase has never been chromatographically separated from the aforementioned (+)-bornyl diphosphate synthase, suggesting that (+)-bornyl diphosphate synthase and (+)-pinene synthase might, in fact, be a single, multifunctional enzyme (McGeady, P., and Croteau, R., Arch. Biochem. Biophys. 317:149-155, 1995). Similarly, the (-)-pinene synthase has never been fully resolved from 1,8-cineole synthase, although, in this case, stereochemical considerations indicate that the two are distinct enzyme species (Croteau et al., Arch. Biochem. Biophys. 309:184-192, 1994; Croteau et al., J. Biol. Chem. 264:2075-2080, 1989).
The unusual ability of the monoterpene synthases to synthesize multiple products from a single substrate requires the nomenclature of these enzymes to be based on the identity of the principal product synthesized by each enzyme. Thus, starting from the common precursor geranyl diphosphate, (+)-bornyl diphosphate synthase characteristically produces a mixture of monoterpenes of which at least 60% is (+)-bornyl diphosphate; 1,8-cineole synthase characteristically produces a mixture of monoterpenes of which at least 60% is 1,8-cineole and (+)-sabinene synthase characteristically produces a mixture of monoterpenes of which at least 60% is (+)-sabinene.